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In situ Digestion of Wheat Cell-wall Polysaccharides
小麦细胞壁多糖的原位消化   

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参见作者原研究论文

本实验方案简略版
Journal of Experimental Botany
May 2014

Abstract

Cell walls of the wheat endosperm are mostly composed of arabinoxylans (AX) and mixed (1→3), (1→4)-β-glucans (BG) (Saulnier et al., 2012). Here, we present an optimized protocol to degrade enzymatically these cell-wall polysaccharides into oligosaccharides, directly from wheat grain cross sections. The main difficulty is to provide a sufficient amount of humidity for the enzyme to be active, while the amount of liquid at the surface of the tissue should stay low to prevent any delocalization of the released products. With this protocol, enzymatic degradation was shown to be efficient and delocalization of released oligosaccharides was estimated below 50 µm (Veličković et al., 2014).

Although it can be employed for other purposes, this in situ enzymatic digestion was primarily developed to obtain molecular images of the cross-sections of wheat endosperm by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Veličković et al., 2014). The cell wall polysaccharides are heterogeneous in structure, exhibit high masses and are entangled into complex networks. Thus, they are not amenable to direct analysis by mass spectrometry and they need to be degraded into smaller compounds as a first step. In this protocol, additional steps corresponding to the deposition of the MALDI matrix are also described.

Keywords: Enzyme application (酶的应用), Arabinoxylans (阿拉伯木聚糖), Beta glucans (β-葡聚), MALDI MSI (MALDI MSI), Wheat section prepartaion (小麦条prepartaion)

Materials and Reagents

  1. Milli-Q quality water (dH2O)
  2. Ethanol (Sigma-Aldrich, catalog number: 02854 )
  3. Acetonitrile (Sigma-Aldrich, catalog number: 271004 )
  4. K2SO4 (Merck KGaA, catalog number: 5153 )
  5. NaCl (Merck KGaA, catalog number: 106404.1 )
  6. CaCl2.2H2O (CARLO ERBA Reagents, catalog number: 433381 )
  7. NaN3 (Merck KGaA, catalog number: 822335 )
  8. NaOH (Sigma-Aldrich, catalog number: 221465 )
  9. H3PO4 (Sigma-Aldrich, catalog number: P6560 )

    Enzymes
  10. α-Amylase from porcine pancreas (Sigma-Aldrich, catalog number: A3176 )
  11. Xylanase M1 from Trichoderma viride (Megazyme International, catalog number: E-XYTRI )
  12. Lichenase [endo-1,3(4)-β-D-glucanase] from Bacillus sp. (Megazyme International, catalog number: E-LICHN )

    MALDI matrix preparation
  13. 2,5-dihydroxybenzoic acid (DHB) (Sigma-Aldrich, catalog number: 85707 )
  14. N,N-dimethylaniline (DMA) (analytical reagent grade) (Thermo Fisher Scientific, catalog number: 121-69-7 )

    Buffers and media
  15. 70% ethanol (see Recipes)
  16. 0.1 M NaOH (see Recipes)
  17. 25 mM CaCl2 (see Recipes)
  18. Buffer for α-Amylase (see Recipes)
  19. 1 mg/ml α-Amylase (see Recipes)
  20. 460 U/ml Xylanase M1 (see Recipes)
  21. 4.6 U/ml Xylanase M1 (see Recipes)
  22. 200 U/ml Lichenase (see Recipes)
  23. 2 U/ml Lichenase (see Recipes)
  24. Saturated K2SO4 at 40 °C (see Recipes)
  25. 50% acetonitrile (see Recipes)
  26. MALDI matrix preparation (see Recipes)

Equipment

  1. Vibratome (MICROM, model: HM 650 V )
  2. In-house designed spraying robot
    Note: Robot was built by adapting an electro-spray probe dismounted from a LCQ Advantage mass spectrometer (Thermo Fisher Scientific) to an X, Y, Z robotic arm (FISNAR, F4300N) (see Figure 1) (see Note 2).


    Figure 1. In-house designed spraying robot for enzyme application. The right hand panel represents the enlarged view of the dashed rectangle zone depicted on the left picture.

  3. Syringe pump infusion 0.2 µl/h to 500 ml/h (Thermo Fischer Scientific, catalog number: 12486350 )
  4. 500 µl glass syringe (Hamilton, model: Gastight 1750 )
  5. Thermo-shaker (Eppendorf, Thermomixer compact)
  6. Incubator (THERMOSI, model: SR 300 )
  7. Home-designed chamber for incubation
    Note: This consists of a rubber gasket sealed glass container (the jar with lid, KORKEN, IKEA of Sweden. Diameter 11 cm; high 10.5 cm, volume 0.5 L) in which is placed a 50 ml glass beaker and a set of weights (e.g. pieces of stones) (see Figure 2). Set of weights are required to keep the glass beaker from floating.


    Figure 2. Schematic representation of home-designed chamber for incubation

  8. pH meter (pHenomenal®, model: pH 1000 L , equipped with a pHenomenal® 221 electrode)

    Other equipment
  9. Glue (Loctite Super Glue Ultra Gel)
  10. Adhesive carbon tape (8 mm x 20 m) (Agar Scientific, catalog number: AGG3939 )
  11. Indium tin oxide (ITO) glass slides (Bruker, catalog number: 237001 ) (see Note 3)
  12. Art paint brush (approximate diameter of the brush: 3-4 mm)
  13. Razor blade (Gillette, Bleue extra)
  14. Petri dish (90 mm x 14.2 mm) (Thermo Fischer Scientific, catalog number: 5184E )
  15. Whatman filter paper (90 mm diameter)
  16. 1.5 ml Eppendorf tube (Eppendorf Safe-Lock quality)
  17. 2 ml plastic Pasteur pipette (Thermo Fischer Scientific, catalog number: 13984 )
  18. Kimwipes paper (Kimberly-Clark)

    Additional, required for MALDI imaging experiments
  19. ImagePrep nebulizing robot (Bruker Daltonics) (see Figure 3)


    Figure 3. ImagePrep nebulizing robot

  20. MTP Slide adapter II (Bruker Daltonics, catalog number: 235380 ) (see Note 4)

Procedure

  1. Rehydration of wheat grains (Philippe et al., 2006).
    1. The germ part (see Figure 4) is removed with a razor blade, perpendicular to the crease zone (longitudinal axis) of the grain, and is discarded.


      Figure 4. Wheat grain and position of razor blade during wheat germ removal. Both sides of grain are presented to easily localize the germ, brush and crease regions.

    2. The grain is then placed in a Petri dish on top of Whatman filter paper moistened with dH2O, and left at +4 °C for 24 h.
      Note: Whole rehydration procedure is skipped for young grains, which are naturally hydrated.
  2. Preparing 70 µm-thin cross sections of wheat grains.
    1. This step is done by using a Vibratome instrument according to instruction provided in the Instruction manual (Thermo Fisher Scientific, 2009). The grain is fixed with some glue onto the magnetic plate of the Vibratome. Orientation of the grain should be such that the longitudinal axis of the grain is placed perpendicular to the magnetic plate. Settings of the Vibratome instrument are as follows: frequency: 60; amplitude: 1.2; speed: 12.
    2. A series of 400 µm-thick slices are cut and discarded, until the surface of the grain becomes flat and parallel to the razor blade of the Vibratome instrument.
    3. Thin sections (approximately 70 µm) are then cut consecutively and placed immediately into Eppendorf tubes filled with 70% ethanol. They are stored at 4 °C until further processing.
  3. Removing starch.
    1. The wheat cross section is grabbed using a paint brush and transferred into a clean Eppendorf tube, filled with 0.5 ml of 1 mg/ml α-Amylase solution. This is allowed to incubate at 40 °C for 24 h on a Thermo-shaker.
    2. Rinse thoroughly by transferring the wheat cross section into a clean Eppendorf tube filled with 1 ml of dH2O. Repeat this step twice. Wash the brush in water after each sample transfer.
      Note: This step is intended to avoid any hydrolysis of starch by endogenous enzyme activity, which would produce some glucans of similar masses as those expected from the hydrolysis of cell walls BG (additionally, when MALDI mass spectrometry is used for further analysis of the tissue, starch generates a strong suppressive effect on the signal).
  4. Placing the cross-section onto an ITO glass plate (see Note 5).
    1. Prepare the ITO glass plate that will receive the cross section:
      1. Place a small piece of adhesive carbon tape at the location where the cross section will be deposited on the ITO glass plate;
      2. With a Pasteur pipette, put a droplet of water on the tape.
    2. Then, with a paint brush, grab the cross section from the Eppendorf tube. Lean the cross section at the surface of the water droplet, allowing it to detach from the paint brush. Proceed very gently, so to avoid any disruption or any distortion of the cross section (Figure 5).


      Figure 5. Positioning of grain section during wheat section grabbing a) and deposition onto the ITO plate (b, c).

      Gently remove excess water with a Kimwipes tissue. Allow sample to dry at RT (approximately 30 min) (see Figure 6).


      Figure 6. Appearance of the ITO plate with mounted wheat sections on it

    3. Mount the ITO glass plate onto the MTP Slide Adapter II (Bruker) according to the manufacturer instructions [available in Bruker, (2012)].

  5. Application of the hydrolytic enzyme on the cross section.
    1. This step uses a homemade robot (see Figure 1 and Note 2). The aim is to deliver a controlled volume of enzyme onto the tissue by using an airbrush device, so that fine droplets of enzyme are applied on the tissue, thereby limiting the diffusion of the oligosaccharides released upon digestion over the tissue.
    2. Spraying of the enzyme is achieved by connecting the electrospray probe mounted on the robotic arm to a syringe pump delivering the enzymatic solution at a constant flow rate of 600 μl/h.
      1. Spraying is assisted pneumatically with nitrogen (1.5 x 105 Pa).
      2. The distance between the needle tip of the Electrospray probe and the ITO plate is 3 cm (Z-axis).
      3. A X, Y deposition pattern following a “brush rectangle” is used (Figure 7).
      4. The movement speed of the robot head was set at 5 mm/s.
      5. Other parameters (X and Y axis start and end coordinates, volume of the enzyme placed into the syringe, number of spraying cycles) are set to ensure that the robot consistently deposits 0.3 μl of enzyme (4.6 U/ml endo-1,4-β-xylanase or 2 U/ml lichenase) per mm2 of sprayed area (corresponding to 0.0014 U xylanase and 0.0006 U lichenase per mm2 tissue).
        Enzymes can be applied individually, consecutively or in mixture.
        Note: Endo-1,4-β-xylanase is used for AX hydrolysis, while lichenase is used for mixed-linkage BG hydrolysis.


        Figure 7. “Brush rectangle” pattern used for enzyme deposition. X and Y “start” and “end” positions are determined from the coordinates of the surface which must be covered by the enzyme.

  6. Incubation.
    1. The wheat cross sections covered by the enzyme in the previous step are allowed to incubate at 40 °C for 4 h.
    2. To prevent liquid from evaporating too fast and the enzyme becomes inactive, a wet atmosphere is maintained by placing the ITO plate into a sealed incubation chamber filled with 150 ml saturated K2SO4 and pre-incubated at 40 °C.
    3. The ITO plate is placed on top of a 50 ml glass beaker weighted with stones and installed into the glass incubator (Figure 2).
    4. After incubation, remove the tissues from the incubation chamber and let dry in the open air (approximately 15 min).
  7. Additional step for MALDI MS measurement of the hydrolyzed cross sections: Deposition of the DMA-DHB MALDI matrix.
    1. This step is performed with an automatic vibration vaporization system from Bruker (ImagePrep, see Figure 3) according to the instructions provided by the manufacturer (Bruker Daltonik, 2007).
    2. The settings are as follows (see Note 6): 1st Phase: 15 cycles: 12% spray power; 20% spray modulation; 2 sec spray time; 15 sec incubation time; 30 sec dry time. 2nd Phase: 40 cycles: 20% spray power; 25% spray modulation; 2 sec spray time; 30 sec incubation time; 60 sec dry time. During whole procedure N2 flow is provided at 2 x 105 Pa.

Representative data

  1. Figure 8 gives a representative example of the type of results which can be expected by this method. The localization of released AX oligosaccharides after in-tissue hydrolysis by endo-1,4-β-xylanase is depicted, as measured by MALDI MS imaging.


    Figure 8. MALDI MS imaging of released AX oligosaccharides at the wheat cross section after endo-1,4-β-xylanase in-situ hydrolysis. Red pixels indicate places where feruloylated AX of DP5 (degree of polymerization 5) is present and green pixels indicate non-feruloylated AX oligosaccharides of DP5 and DP6.

Recipes

  1. 70% ethanol
    Place 700 ml ethanol in a glass bottle and complete to 1,000 ml with dH2O
  2. 0.1 M NaOH
    Weigh 0.4 g NaOH and dissolve in 100 ml of dH2O
  3. 25 mM CaCl2
    Weigh 0.018 g CaCl2.2H2O and dissolve in 5 ml of dH2O
  4. Buffer for α-Amylase (20 mM Na-phosphate buffer with 2 mM NaCl and 0.25 mM CaCl2, pH 6.9 with 0.02% NaN3 as stabilizer)
    Place 450 ml of dH2O in a glass bottle, and add
    1.15 g H3PO4
    0.058 g NaCl
    5 ml of 25mM CaCl2
    Mix thoroughly and adjust pH to 6.9 with 0.1 M NaOH
    Complete to 500 ml with dH2O
    Add 0.1 g NaN3
    Stored at +4 °C
  5. One mg/ml α-Amylase
    Dissolve 10 mg of α-Amylase in 10 ml of buffer for α-Amylase
  6. 460 U/ml Xylanase M1
    Place 80 μl of dH2O in a clean Eppendorf tube
    Add 20 μl of manufacture stock of Xylanase M1 (2,300 U/ml)
    Gently shake before sampling
  7. 4.6 U/ml xylanase M1
    Place 990 μl of dH2O in a clean Eppendorf tube
    Add 10 μl of 460U/ml Xylanase M1
  8. 200 U/ml lichenase
    Place 80 μl of dH2O in a clean Eppendorf tube.
    Add 20 μl of manufacture stock of lichenase (1,000 U/ml)
    Gently shake before sampling
  9. 2 U/ml lichenase
    Place 990 μl of dH2O in a clean Eppendorf tube
    Add 10 μl of 200 U/ml lichenase
  10. Saturated K2SO4 at 40 °C
    Weigh 75 g K2SO4 and dissolve in 500 ml of dH2O (warm up to 40 °C with stirring until complete dissolution)
  11. 50% acetonitrile
    Place 250 ml of acetonitrile in a glass bottle
    Complete to 500 ml with dH2O
  12. MALDI matrix preparation
    Weigh 500 mg DHB
    Dissolve in 5 ml of 50% acetonitrile
    Add 100 μl of DMA
    Careful: DMA is toxic in contact with skin and suspected of causing cancer. Wear protective gloves, protective clothing, eye protection and face protection. Proceed under a fume hood.

Notes

  1. This protocol was performed on mature wheat grains (700 D) from several cultivars of the genus Triticum aestivum: Recital, Malacca, Virtuose, Magdalena, Crousty, Thesee, Baltimore, Sisley, Aligre and Tamaro.
    The same protocol was also performed on young stages of development (245 D) from seeds of the cultivar Recital.
  2. The basic idea of the home-made robot is to put an airbrush-type device on a X, Y, Z robotic arm, and be able to control the liquid flow rate through the airbrush. To do so, we have dismounted an Electrospray probe from an old LCQ Advantage mass spectrometer and adjusted this probe to a FISNAR 4300N robot. Flow rate through the probe is controlled by a syringe pump, delivering a typical flow rate of 1-100 µl/min. Nitrogen is used as a co-axial nebulizing gas, at a pressure of 1.5 x 105 Pa.
    The FISNAR 4300N robot is controlled by a Teach Pendant device, which enables to program the motion patterns for the enzyme deposition. A program consists in defining the start and end positions (defined by X, Y, Z coordinates), the pattern and the speed of the robotic arm movement. “Brush rectangle” pattern is one of the available patterns and is the one that we used in our experiments. It is depicted in Figure 7. The programing and use of the FISNAR 4300N robot is very well explained in (FISNAR, 2012).
  3. ITO glass plates: These plates exhibit a conductive side (coated by Indium Tin Oxide). The use of these conductive plates was imposed in our case by the MALDI mass spectrometry measurements that were subsequently made on the hydrolyzed tissues. However, any glass plate with the same dimensions (75 x 25 x 0.9 mm) can be used in principle.
  4. Adapter for glass slides (MTP Slide adapter II for glass slides): This adapter is provided by Bruker in order to introduce ITO glass plates into an Autoflex III MALDI mass spectrometer. It was used herein as a convenient holder for all the steps performed with the homemade spraying robot. However, any rectangular holder of the same dimensions (12.8 x 8.8 cm) can be used instead.
  5. Note that the most critical step for maintaining reproducibility of in-tissue digestion is transferring the tissue at the ITO plate (Procedure 4). It must be very carefully performed to avoid any damage of the tissue cell wall network. Several sections (3 to 4) must usually be deposited so to ensure that at least one of these is of good quality (this is also dependent of the wheat cultivar and stage of development of the seed. There is no other trick than experience!). Application of the enzyme is performed automatically by the robot so the reproducibility of this step is high (does not depend on the position of the section on the plate). It is however recommended that a fresh enzyme solution is prepared for this step. Our experience is that these points are the main reason of result variance (which was estimated to be of 14% with our detection method based on MALDI MS).
  6. The ImagePrep device produces an aerosol by vaporization of the MALDI matrix through a vibration blade, i.e. a thin sheet of stainless steel with pinholes. These holes can get clogged or expand after several uses. The settings (spray power, e.g.) can be adjusted by the operator to ensure a similar coating of the tissue with the MALDI matrix, even though the blade has been used several times. However, we recommend replacing the vibrational blade after ten uses (or whenever it looks damaged) to ensure reproducible application of the MALDI matrix.

Acknowledgments

This work was supported in part through a postdoctoral fellowship (Dušan Veličković) from INRA (Institut National de Recherche Agronomique, France) and AgreenSkills. We are very grateful to Fabienne Guillon and Luc Saulnier (INRA Biopolymers, Interaction, Assemblies, Nantes, France) for helpful discussions about the enzymatic hydrolysis of wheat cell walls. This protocol is a modified version of the protocol previously described in Veličković et al. (2014).

References

  1. Bruker Daltonic (2007). Image Prep User Manual.
  2. Bruker Daltonic (2012). Instructions for Use Tools for MALDI Imaging.
  3. FISNAR (2012). F4000N Series Robot Operating Manual.
  4. Philippe, S., Robert, P., Barron, C., Saulnier, L. and Guillon, F. (2006). Deposition of cell wall polysaccharides in wheat endosperm during grain development: Fourier transform-infrared microspectroscopy study. J Agric Food Chem 54(6): 2303-2308.
  5. Saulnier, L., Guillon, F. and Chateigner-Boutin, A. L. (2012). Cell wall deposition and metabolism in wheat grain. J Cereal Sci 56(1): 91-108.
  6. Thermo Fisher Scientific (2009). Thermo Scientific Microtome with vibrating blade MICROM HM 650V: Instruction manual.
  7. Veličković, D., Ropartz, D., Guillon, F., Saulnier, L. and Rogniaux, H. (2014). New insights into the structural and spatial variability of cell-wall polysaccharides during wheat grain development, as revealed through MALDI mass spectrometry imaging. J Exp Bot 65(8): 2079-2091.

简介

小麦胚乳的细胞壁主要由阿拉伯木聚糖(AX)和混合的(1→3),(1→4)-β-葡聚糖(BG)组成(Saulnier等人,2012)。在这里,我们提出一个优化的协议,以酶法将这些细胞壁多糖降解为低聚糖,直接从小麦籽粒横截面。主要的困难是提供足够量的湿度以使酶活性,而组织表面处的液体量应保持低以防止释放的产物的任何离域。使用该方案,酶降解显示是有效的,并且释放的寡糖的离域化估计低于50μm(Veličkovic等人,2014)。虽然它可以用于其他目的,但是这种原位酶消化主要用于通过基质辅助激光解吸/电离(MALDI)获得小麦胚乳的横截面的分子图像)质谱(Veličkovic等人,2014)。细胞壁多糖在结构上是异质的,表现出高质量并且缠结到复杂网络中。因此,它们不适合通过质谱法直接分析,并且它们需要作为第一步骤降解为较小的化合物。在该方案中,还描述了对应于MALDI基质的沉积的另外的步骤。

关键字:酶的应用, 阿拉伯木聚糖, β-葡聚, MALDI MSI, 小麦条prepartaion

材料和试剂

  1. Milli-Q质量水(dH 2 O)
  2. 乙醇(Sigma-Aldrich,目录号:02854)
  3. 乙腈(Sigma-Aldrich,目录号:271004)

  4. (Merck KGaA,目录号:5153)
  5. NaCl(Merck KGaA,目录号:106404.1)

  6. (Carlo ERBA Reagents,目录号:433381)
  7. NaN 3(Merck KGaA,目录号:822335)
  8. NaOH(Sigma-Aldrich,目录号:221465)
  9. H sub 3 PO 4(Sigma-Aldrich,目录号:P6560)


  10. 来自猪胰腺的α-淀粉酶(Sigma-Aldrich,目录号:A3176)
  11. 来自绿色木霉的木聚糖酶M1(Megazyme International,目录号:E-XYTRI)
  12. 来自芽孢杆菌属(Megazyme International,目录号:E-LICHN)的lichenase [内切-1,3(4)-β-D-葡聚糖酶]

    MALDI基质制备
  13. 2,5-二羟基苯甲酸(DHB)(Sigma-Aldrich,目录号:85707)
  14. N,N-二甲基苯胺(DMA)(分析试剂级)(Thermo Fisher Scientific,目录号:121-69-7)

    缓冲区和媒体
  15. 70%乙醇(见配方)
  16. 0.1 M NaOH(见配方)
  17. 25mM CaCl 2(参见配方)
  18. α-淀粉酶的缓冲液(参见配方)
  19. 1mg/mlα-淀粉酶(参见配方)
  20. 460 U/ml木聚糖酶M1(见配方)
  21. 4.6U/ml木聚糖酶M1(参见配方)
  22. 200 U/ml Lichenase(参见配方)
  23. 2 U/ml Lichenase(参见配方)
  24. 40℃时饱和K <2> SO 4(参见配方)
  25. 50%乙腈(见配方)
  26. MALDI基质制备(参见配方)

设备

  1. Vibratome(MICROM,型号:HM 650V)
  2. 内部设计的喷涂机器人
    注意:机器人通过将从LCQ Advantage质谱仪(Thermo Fisher Scientific)卸下的电喷射探针适配到X,Y,Z机器人臂(FISNAR,F4300N)(参见图1)来构建(参见注释2 )。


    图1.内部设计的喷涂机器人用于酶应用。 右侧面板代表左图所示的虚线矩形区域的放大视图。

  3. 注射泵输注0.2μl/h至500ml/h(Thermo Fischer Scientific,目录号:12486350)
  4. 500μl玻璃注射器(Hamilton,型号:Gastight 1750)
  5. 热振荡器(Eppendorf,Thermomixer compact)
  6. 孵化器(THERMOSI,型号:SR 300)
  7. 家庭设计的孵化室
    注意:这包括橡胶垫圈密封的玻璃容器(具有盖的罐子,KORKEN,IKEA of Sweden,直径11cm;高10.5cm,体积0.5L),其中放置了50ml玻璃烧杯和一套 的重量(例如石块)(见图2)。 需要一组重量以保持玻璃杯不漂浮。


    图2.家庭设计的孵化室示意图

  8. pH计(pHenomenal ,型号:pH 1000L,配备pHenomenal 221电极)

    其他设备
  9. 胶水(Loctite Super Glue Ultra Gel)
  10. 粘性碳带(8mm×20μm)(Agar Scientific,目录号:AGG3939)
  11. 氧化铟锡(ITO)载玻片(Bruker,目录号:237001)(参见注释3)
  12. 艺术漆刷(大约直径:3-4毫米)
  13. 剃刀刀片(Gillette,Bleue extra)
  14. 培养皿(90mm×14.2mm)(Thermo Fischer Scientific,目录号:5184E)
  15. Whatman滤纸(直径90mm)
  16. 1.5 ml Eppendorf管(Eppendorf安全锁质量)
  17. 2ml塑料巴斯德吸管(Thermo Fischer Scientific,目录号:13984)
  18. Kimwipes纸(Kimberly-Clark)

    额外的,MALDI成像实验所需的
  19. ImagePrep雾化机器人(Bruker Daltonics)(见图3)


    图3. ImagePrep雾化机器人

  20. MTP滑动适配器II(Bruker Daltonics,目录号:235380)(参见注释4)

程序

  1. 小麦籽粒的再水化(Philippe等人,2006)。
    1. 胚芽部分(参见图4)用剃刀刀片移除, 垂直于颗粒的折痕区(纵轴),和 被丢弃。


      图4.小麦籽粒和剃刀刀片的位置 在小麦胚芽去除期间。粮食的两面都很容易呈现 定位胚芽,刷子和褶皱区域
    2. 然后将谷粒放置在用dH 2 O润湿的Whatman滤纸上的培养皿中,并在+ 4℃下放置24小时。
      注意:对于年轻的谷物,其被自然水合,所以完全水化程序被跳过
  2. 制备70μm薄的小麦籽粒横截面。
    1. 该步骤通过使用根据的Vibratome仪器进行 指导手册(Thermo Fisher 科学,2009)。 颗粒用一些胶固定在磁体上 板的Vibratome。 颗粒的取向应该是这样的   晶粒的纵轴垂直于磁体放置 盘子。 Vibratome仪器的设置如下:频率: 60; 振幅:1.2; 速度:12.
    2. 一系列400微米厚的切片 切割并丢弃,直到谷物的表面变平 平行于Vibratome仪器的剃刀刀片。
    3. 瘦 (大约70μm),然后放置 立即加入装有70%乙醇的Eppendorf管中。 他们是 储存在4℃直至进一步处理。
  3. 去除淀粉。
    1. 使用油漆刷抓住小麦横截面并转移 加入到装有0.5ml 1mg/mlα-淀粉酶的干净的Eppendorf管中 解。 允许将其在40℃下孵育24小时 热振荡器。
    2. 通过转移小麦十字架彻底冲洗 切片放入装有1ml dH 2 O的干净的Eppendorf管中。 重复 这一步两次。 每次样品转移后在水中清洗刷子。
      注意:此步骤旨在避免淀粉的任何水解 内源酶活性,其将产生一些类似的葡聚糖 如从细胞壁BG的水解预期的那些 (另外,当使用MALDI质谱法进行进一步分析时   的组织,淀粉对其产生强烈的抑制作用 信号)。
  4. 将横截面放置在ITO玻璃板上(见注5)
    1. 准备接收横截面的ITO玻璃板:
      1. 放置一小块胶带在胶带的位置 横截面将沉积在ITO玻璃板上;
      2. 用巴斯德吸管,将一滴水放在磁带上。
    2. 然后,用油漆刷,抓住Eppendorf的横截面 管。 倾斜水滴表面的横截面, 允许其从漆刷分离。 继续非常轻柔,所以 避免任何中断或横截面的任何变形(图5)。


      图5.小麦部分抓取a)和沉积到ITO板(b,c)期间的颗粒截面的定位。

      用Kimwipes组织轻轻取出多余的水。 使样品在室温下干燥(约30分钟)(见图6)

      图6.其上带有小麦部分的ITO板的外观

    3. 将ITO玻璃板安装到MTP Slide Adapter II(Bruker) 根据制造商的说明[可得自Bruker, (2012)]。

  5. 水解酶在横截面上的应用。
    1. 此步骤使用自制机器人(见图1和注2)。 目的是   通过使用将可控体积的酶递送到组织上 喷枪装置,使得细小的酶滴施加在其上 组织,从而限制释放的寡糖的扩散 在组织上消化。
    2. 喷洒酶 通过连接安装在机器人臂上的电喷雾探针实现    到以恒定流量递送酶溶液的注射泵   速率600μl/h。
      1. 用氮气(1.5×10 5 Pa)气动辅助喷雾。
      2. 电喷雾探针的针尖与ITO板之间的距离为3cm(Z轴)。
      3. 使用"刷矩形"后的X,Y沉积图案(图7)。
      4. 机器人头的移动速度设定为5mm/s。
      5. 其他参数(X和Y轴起点和终点坐标,体积 放入注射器中的酶,喷雾周期数) 以确保机器人一致地沉积0.3微升的酶(4.6 U/ml内切-1,4-β-木聚糖酶或2U/ml地衣多糖酶)/mm 2喷雾面积 (对应于0.0014U木聚糖酶和0.0006U淀粉酶/mm 2组织)。
        酶可以单独,连续或混合使用。
        注意:内切-1,4-β-木聚糖酶用于AX水解,而地衣多糖酶用于混合连接BG水解。


        图7.用于酶沉积的"刷矩形"图案。 X和Y.  根据坐标确定"开始"和"结束"位置 必须被酶覆盖的表面。

  6. 孵化。
    1. 将前述步骤中酶覆盖的小麦横截面在40℃下孵育4小时。
    2. 为了防止液体蒸发太快和酶变成 非活性的,通过将ITO板放入a中来保持湿气氛   密封的孵育室,其填充有150ml饱和K 2 SO 4和NH 4 在40℃预温育。
    3. 将ITO板放置在50的顶部 ml玻璃烧杯用石头称重并安装到玻璃 孵化器(图2)。
    4. 孵育后,从孵化室中取出组织,并在空气中干燥(约15分钟)。
  7. 用于MALDI MS测量水解截面的附加步骤:沉积DMA-DHB MALDI基质。
    1. 该步骤用自动振动蒸发系统进行 从Bruker(ImagePrep,参见图3) 由制造商提供(Bruker Daltonik,2007)。
    2. 的 设置如下(参见注释6):1 st 相:15个循环:12%喷雾 功率; 20%喷雾调制; 2秒喷涂时间; 15秒孵育时间; 30秒干燥时间。 2个阶段:40个循环:20%喷雾功率; 25%喷雾 调制; 2秒喷涂时间; 30秒孵育时间; 60秒干燥时间。 在整个过程期间,在2×10 5 Pa下提供N 2流。

代表数据

  1. 图8给出了可以通过该方法预期的结果类型的代表性实例。如通过MALDI MS成像所测量的,描绘了通过内-1,4-β-木聚糖酶在组织内水解后释放的AX寡糖的定位。


    图8.内切-1,4-β-木聚糖酶原位水解后小麦横截面上释放的AX寡糖的MALDI MS成像。红色像素表示DP5的阿魏酰化AX(聚合度)的位置5) 存在和绿色像素表示DP5和DP6的非阿魏酸化AX寡糖。

食谱

  1. 70%乙醇
    将700ml乙醇置于玻璃瓶中,用dH 2 O/dm 2完成至1000ml
  2. 0.1 M NaOH
    称重0.4g NaOH并溶解在100ml dH 2 O中
  3. 25mM CaCl 2。 称取0.018g CaCl 2·2H 2·2H 2 O并溶解在5ml的dH 2 O中。
  4. α-淀粉酶的缓冲液(具有2mM NaCl和0.25mM CaCl 2的20mM磷酸钠缓冲液,pH 6.9,含有0.02%NaN 3作为稳定剂) 将450ml dH 2 O置于玻璃瓶中,并加入
    1.15g H sub 3 PO 4 4/
    0.058g NaCl
    5ml 25mM CaCl 2·6H 2 O 充分混合并用0.1M NaOH将pH调节至6.9 用dH 2 O完全达到500ml
    加入0.1g NaN 3
    储存在+4°C
  5. 1mg/mlα-淀粉酶 将10毫克α-淀粉酶溶解在10毫升α-淀粉酶缓冲液中
  6. 460U/ml木聚糖酶M1 / 将80μldH 2 O置于干净的Eppendorf管中
    加入20μl的木聚糖酶M1(2,300 U/ml)的生产库存
    在取样前轻轻摇动
  7. 4.6U/ml木聚糖酶M1 将990μldH 2 O置于干净的Eppendorf管中
    加入10μl460U/ml木聚糖酶M1 /
  8. 200U/ml地衣淀粉酶 放置80微升的dH 2 O在干净的Eppendorf管中。
    加入20μl地衣霉素(1,000 U/ml)的生产库存
    在取样前轻轻摇动
  9. 2 U/ml地衣淀粉酶 将990μldH 2 O置于干净的Eppendorf管中
    加入10μl的200 U/ml lichenase
  10. 在40℃下的饱和K 2 SO 4 称取75g K 2 SO 4并溶于500ml dH 2 O(温热至40℃,搅拌直至完全溶解)
  11. 50%乙腈 将250 ml乙腈放入玻璃瓶中
    用dH 2 O完全达到500ml
  12. MALDI基质制备
    称重500 mg DHB
    溶于5ml 50%乙腈中 加入100μlDMA
    注意:DMA与皮肤接触并有怀疑致癌的毒性。 穿戴防护手套,防护服,眼睛防护和面部防护。 在通风橱下进行。

笔记

  1. 该方案在来自普通小麦属的多个品种的成熟小麦谷粒(700D)上进行:Recital,Malacca,Virtuose,Magdalena,Crousty,Thesee,Baltimore,Sisley,Aligre和Tamaro。
    同样的方案也在年轻的发育阶段(245D)从栽培品种Recital的种子进行
  2. 自制机器人的基本思想是将喷枪型装置放置在X,Y,Z机器人手臂上,并且能够控制通过喷枪的液体流速。为此,我们从旧的LCQ Advantage质谱仪上拆下了Electrospray探针,并将该探针调整为FISNAR 4300N机器人。通过探针的流速由注射泵控制,递送典型的流速为1-100μl/min。氮气用作同轴雾化气体,压力为1.5×10 5 Pa。
    FISNAR 4300N机器人由示教器(Teach Pendant)控制,可以对酶沉积的运动模式进行编程。程序包括定义开始和结束位置(由X,Y,Z坐标定义),机器人手臂运动的模式和速度。 "刷矩形"模式是可用模式之一,是我们在实验中使用的模式。如图7所示。FISNAR 4300N机器人的编程和使用在(FISNAR,2012)中有很好的解释。
  3. ITO玻璃板:这些板具有导电侧(由氧化铟锡涂布)。在我们的情况下,通过随后在水解组织上进行的MALDI质谱测量来强制使用这些导电板。然而,原则上可以使用具有相同尺寸(75×25×0.9mm)的任何玻璃板
  4. 玻璃载玻片适配器(MTP载玻片适配器II):该适配器由Bruker提供,以将ITO玻璃板引入Autoflex III MALDI质谱仪。其在本文中用作用于使用自制喷洒机器人执行的所有步骤的方便的保持器。但是,可以使用相同尺寸(12.8 x 8.8厘米)的任何矩形支架。
  5. 注意,保持组织内消化的可重复性的最关键的步骤是在ITO板上转移组织(过程4)。它必须非常仔细地进行以避免组织细胞壁网络的任何损伤。几个部分(3到4)通常必须沉积,以确保至少其中一个是良好的质量(这也取决于小麦品种和种子的发展阶段。没有其他的骗子而不是经验! 。酶的应用由机器人自动进行,因此该步骤的再现性高(不依赖于板上的部分的位置)。然而,建议为该步骤制备新鲜的酶溶液。我们的经验是这些点是结果方差的主要原因(据我们基于MALDI MS的检测方法估计其为14%)。
  6. ImagePrep装置通过振动叶片(即,具有针孔的不锈钢薄片)蒸发MALDI基质产生气溶胶。这些孔在几次使用后可能堵塞或膨胀。可以由操作者调节设置(喷射功率,例如),以确保组织与MALDI基质的类似涂层,即使刀片已经使用多次。然而,我们建议在十次使用后(或每当它看起来损坏时)更换振动刀片,以确保MALDI基质的可重复应用。

致谢

这项工作部分通过来自INRA(法国国家农业研究所)和AgreenSkills的博士后研究金(DušanVeličkovic)得到支持。我们非常感谢Fabienne Guillon和Luc Saulnier(INRA Biopolymers,Interaction,Assemblies,Nantes,法国)对小麦细胞壁酶水解的有益讨论。该协议是先前在Veličkovic等人(2014)中描述的协议的修改版本。

参考文献

  1. Bruker Daltonic(2007)。 图像准备用户手册。
  2. Bruker Daltonic(2012)。 MALDI Imaging的使用说明。
  3. FISNAR(2012)。 F4000N系列机器人操作手册。
  4. Philippe,S.,Robert,P.,Barron,C.,Saulnier,L.and Guillon,F.(2006)。 在谷物发育过程中在小麦胚乳中沉积细胞壁多糖:傅立叶变换红外显微光谱研究。 a A> J Agric Food Chem 54(6):2303-2308。
  5. Saulnier,L.,Guillon,F。和Chateigner-Boutin,A.L。(2012)。 小麦籽粒中的细胞壁沉积和代谢。谷物科学em(56)(1):91-108。
  6. Thermo Fisher Scientific(2009)。 带有振动刀片的Thermo Scientific Microtome MICROM HM 650V:说明书。
  7. Veličković,D.,Ropartz,D.,Guillon,F.,Saulnier,L.和Rogniaux,H。(2014)。 关于小麦籽粒发育过程中细胞壁多糖的结构和空间变异性的新见解,通过MALDI质谱成像显示。 65(8):2079-2091。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Veličković, D. and Rogniaux, H. (2014). In situ Digestion of Wheat Cell-wall Polysaccharides. Bio-protocol 4(23): e1306. DOI: 10.21769/BioProtoc.1306.
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Theodora Tryfona
University of Cambridge
Hi Dusan, Thank you for your response. Can you also tell me why did you add DMA to your DHB matrix? I would like to use your method on stems can you advice on the critically important steps of the protocol? When you take your optical images do you do it on the same section or different? Do you stain? So sorry I am bombarding you with so many questions. Best wishes, Dora
3/9/2018 1:59:28 AM Reply
Dušan Veličković
Pacific Northwest National Laboratory

Hi Theodora, Thank you for your interest in our protocol! So, DMA/DHB matrix is by far the best MALDI matrix for carbohydrates (sugars and oligossacharides). You can find publication from our lab about this: http://onlinelibrary.wiley.com/doi/10.1002/rcm.5060/abstract There is theory that DMA mimics Schiff base formation with sugars which increase their sensitivity during ionization: http://onlinelibrary.wiley.com/doi/10.1002/rcm.3265/abstract For your second question, I usually scan sample before application of matrix. In this particular case I didn't stain it, but in fact you can stain it after finishing your MALDI run: wash matrix with 70% EtOH (by dipping plate in 70%EtOH solution 3x2min) and then you can stain it. Or you can stain consecutive section if this washing doesn't give you good results. For your stems: I did analyze long time ago xylans from maize stem: be aware that you need to know general chemical organization and polyssacharides composition. For example I did remember that these xylans in maize stem are very very rich in acetylation and feruliation so I needed to remove these decorations by KOH treatment before application of enzymes (since these decorations will hide glycosidic bond that enzyme recognizes). I hope this will guide your experiments. Feel free to contact me if you need further assistance. Dusan

3/9/2018 11:08:22 AM


Theodora Tryfona
University of Cambridge
Can you please explain why at your incubator chamber set up you have used a filter papaer? What is the purpose of that? Also why saturated K2SO4? Thank you.
3/8/2018 8:02:41 AM Reply
Dušan Veličković
Pacific Northwest National Laboratory

Hi Theodora, Thanks for your questions! Filter paper is used to saturate air in chamber with solvent vapor, so that sample doesn't dry there. In fact, filter paper absorbs the liquid in the solvent and provides more surface area for evaporation. More surface area=faster evaporation. More evaporation means more solvent vapor in the chamber's air. A saturated solution of salt in water is used to maintain particular values of relative humidity inside chamber. K2SO4 gives the highest relative humidity. At 45C saturated K2SO4 has 96% relative humidity, which we used in our experiments. We tried also different salts solutions, but every time droplet of enzyme will not "survive" after overnight incubation. With K2SO4 we could incubate 24h without drying of enzyme droplet spotted on the tissue. I hope this will help you in your experiments, Dusan

3/8/2018 11:58:51 AM